The overarching goal of the proposed studies is to preclinically validate transglutaminase 2 (TG2) as a drug target for celiac disease (CeD), using the most advanced small molecule leads and the most advanced animal models available to date. CeD is a life-long, gluten-induced inflammatory disorder of the small intestine for which no non-dietary therapy exists to date. Although TG2 is widely regarded as the most attractive small molecule drug target for CeD therapy, definitive support for this pharmacologic hypothesis remains elusive. In preliminary studies, we have studied a class of irreversible TG2 inhibitors based on the mildly electrophilic 3- bromo-4,5-dihydroisoxazole motif, and have identified a lead compound (ERW1041E) with good in vitro and in vivo activity. Importantly, ERW1041E is the only TG2 inhibitor to our knowledge that has been shown to block TG2 in vivo on a once-daily dosing schedule. Separately, by engineering double and triple transgenic mouse strains, we have established an animal model that shows all four hallmarks of CeD: (i) gluten-dependent T cell activation; (ii) gluten-dependent autoantibody production; (iii) gluten-dependent infiltrationof lymphocytes into the intestinal epithelium; and (iv) gluten-dependent villous atrophy. Last but not least, we have demonstrated that oral gluten exposure activates TG2 in the small intestine of this mouse model and can be blocked by our lead compound ERW1041E. Our plans for validation of TG2 as a therapeutic target for CeD are described under two Specific Aims: 1) We will identify improved analogs of ERW1041E through synthesis and in vitro evaluation of two focused compound series. The most promising analogs will be subjected to pharmacokinetic and preliminary toxicological analysis. In particular, we seek to improve the isoform specificity o ERW1041E without sacrificing its potency, tolerability, oral bioavailability, or solubility. 2) To validate TG2 as a CeD drug target, the pharmacodynamic relationship between inhibitor dose and intestinal TG2 activity will be defined for ERW1041E and up to four improved analogs. From this data, sub-maximal and maximal inhibitory doses of each compound will be selected, and used to interrogate the role of TG2 in different clinically relevant scenarios that can be accurately mimicked in our mouse models. Together, these studies will provide clear direction for clinical evaluation of the most promising drug candidate. If the above studies are successful, then the most promising TG2 inhibitor from this project may be a suitable candidate for testing in CeD patients. Notably, recent efforts have led to the development of a short (2-week), small (40-patient) clinical protocol for such proof-of-concept studies. Thus, pending appropriate non-clinical safety studies, a new target for CeD therapy could be clinically validated within the next 5 years.